Jing-Jun Zhou

The myocardial protective effects of a moderate-potassium adenosine–lidocaine cardioplegia in pediatric cardiac surgery

OBJECTIVES We sought to evaluate a moderate-potassium cardioplegic solution using adenosine and lidocaine as the arresting and protecting cardioprotective combination in pediatric cardiac surgery. METHODS One hundred thirty-four patients with congenital heart disease were randomly allocated to one of 3 groups according to the cardioplegia formula used: the high-potassium (HP) group (K(+), 20 mmol/L), 46 patients; the high-potassium adenosine-lidocaine (HPAL) group (K(+), 20 mmol/L; adenosine, 0.7 mmol/L; and lidocaine, 0.7 mmol/L), 44 patients; and the moderate-potassium adenosine-lidocaine (MPAL) group (K(+), 10 mmol/L; adenosine, 0.7 mmol/L; and lidocaine, 0.7 mmol/L), 44 patients. Hemodynamic data during the operation and postoperative data were recorded. Serum cardiac troponin I concentrations were examined at the time points of before cardiopulmonary bypass and 1, 3, 6, 12, and 24 hours after aortic crossclamp removal. RESULTS At the end of cardiopulmonary bypass and modified ultrafiltration, the systolic and pulse pressures of the MPAL group were significantly increased compared with the respective values of the HP group. At the time points of 1 to 12 hours after reperfusion, the levels of serum cardiac troponin I were significantly decreased in the MPAL group compared with those in the HP and HPAL groups. CONCLUSIONS The MPAL cardioplegia formula was associated with better myocardial protective effects.

Calpain inhibitor MDL 28170 protects against the Ca2+ paradox in rat hearts.

The calcium paradox represents an important model in which to study myocardial injuries due to intracellular Ca2+ overload. In a previous study, calpain was transiently activated in Ca2+‐paradoxic hearts. The aim of the present study was to determine the role of calpain in myocardial dysfunction in hearts subjected to the Ca2+ paradox and to elucidate the underlying mechanisms. Rat hearts were isolated, Langendorff perfused and subjected to the Ca2+ paradox, which was induced by 3 min Ca2+ depletion followed by 30 min Ca2+ repletion, in the presence or absence of the calpain inhibitor 10 umol/L MDL 28170. Cardiac function was evaluated. Furthermore, cell death and the degradation of troponin I (TnI) were assessed and calpain activity was determined by measurement of the α‐fodrin fragment and confocal image analysis. Upon Ca2+ repletion, the hearts immediately deteriorated, exhibiting a marked depression in cardiac function and an enlarged myocardial injury area. This was accompanied by significant increases in lactate dehydrogenase, mitochondrial release of cytochrome c, the apoptotic index and degraded TnI. These changes were significantly inhibited by MDL 28170, with the exception of TnI degradation. Compared with the control group, Ca2+‐paradoxic hearts showed a marked increase in cleaved 150 kDa fragments resulting from specific calpain‐mediated proteolysis of α‐fodrin. This effect was attenuated by MDL 28170. Confocal image analysis revealed the translocation of both μ‐ and m‐calpain to the sarcolemmal membrane in Ca2+‐paradoxic hearts, indicating increased activity of both isoforms. The results suggest that the Ca2+ paradox promotes calpain activity, leading to necrosis, apoptosis and myocardial dysfunction.

The Blockade of Transmembrane Cl⁻ Flux Mitigates I/R-Induced Heart Injury via the Inhibition of Calpain Activity.

Aims: The aim of this study was to determine whether calpain is involved in Cl- -induced myocardial ischemia/reperfusion (I/R) injury. Methods: Isolated rat hearts were subjected to either 45 min of global no-flow ischemia followed by reperfusion or successive perfusion with Ca2+ -free KH solution for 3 min and normal KH solution for 30 min, also known as Ca2+ paradox. Results: The hearts in the I/R group exhibited increases in myocardial injury area, LDH release, caspase 3 activity and apoptotic indices and a marked decline in cardiac performance. As was the case regarding the effects of MDL 28170, an inhibitor of calpain, treatment with 5 µM NPPB, 5 µM DIDS and low Cl- significantly attenuated cardiac injury. Moreover, each of the treatments significantly protected against Ca2+ overload-induced injury in the setting of Ca2+ paradox. The Western blot and immunofluorescence data revealed that there was an increase in the percentages of calpain membrane-positive cells and the numbers of fragments resulting from the calpain-mediated proteolysis of α-fodrin in both the I/R and the Ca2+ paradox, indicating that the activation of calpain occurred. More importantly, these effects were mitigated by the blockade of transmembrane Cl- flux, as was accomplished via MDL 28170. Conclusion: Our results provide evidence that the blockade of transmembrane Cl- flux mitigates I/R-induced cardiac injury via the inhibition of calpain activity. They also indicate that intracellular Ca2+ overload regulates calpain activation in the setting of Cl- -induced injury.

Different Roles for Contracture and Calpain in Calcium Paradox-Induced Heart Injury

The Ca2+ paradox represents a good model to study Ca2+ overload injury in ischemic heart diseases. We and others have demonstrated that contracture and calpain are involved in the Ca2+ paradox-induced injury. This study aimed to elucidate their roles in this model. The Ca2+ paradox was elicited by perfusing isolated rat hearts with Ca2+-free KH media for 3 min or 5 min followed by 30 min of Ca2+ repletion. The LVDP was measured to reflect contractile function, and the LVEDP was measured to indicate contracture. TTC staining and the quantification of LDH release were used to define cell death. Calpain activity and troponin I release were measured after Ca2+ repletion. Ca2+ repletion of the once 3-min Ca2+ depleted hearts resulted in almost no viable tissues and the disappearance of contractile function. Compared to the effects of the calpain inhibitor MDL28170, KB-R7943, an inhibitor of the Na+/Ca2+ exchanger, reduced the LVEDP level to a greater extent, which was well correlated with improved contractile function recovery and tissue survival. The depletion of Ca2+ for 5 min had the same effects on injury as the 3-min Ca2+ depletion, except that the LVEDP in the 5-min Ca2+ depletion group was lower than the level in the 3-min Ca2+ depletion group. KB-R7943 failed to reduce the level of LVEDP, with no improvement in the LVDP recovery in the hearts subjected to the 5-min Ca2+ depletion treatment; however, KB-R7943 preserved its protective effects in surviving tissue. Both KB-R7943 and MDL28170 attenuated the Ca2+ repletion-induced increase in calpain activity in 3 min or 5 min Ca2+ depleted hearts. However, only KB-R7943 reduced the release of troponin I from the Ca2+ paradoxic heart. These results provide evidence suggesting that contracture is the main cause for contractile dysfunction, while activation of calpain mediates cell death in the Ca2+ paradox.

Protective effects of adenosine on the diabetic myocardium against ischemia-reperfusion injury: role of calpain.

Myocardial ischemia/reperfusion injury (I/RI) is the principal cause of mortality and morbidity in diabetic patients undergoing cardiac surgery. However, there is no specific measure available to protect diabetic hearts in this clinical setting. Our clinical studies showed that adenosine pre-treatment or post-treatment and adding adenosine to cardioplegia solution had significant myocardial protective effects in patients undergoing cardiac surgery. However, the specific protective effects and mechanisms of adenosine in diabetic myocardial I/RI are not clear. Calpain is an important proteolytic enzyme in the myocardium. Studies show that the activation of calpain is an injury factor in not only the diabetic myocardium but also myocardial I/RI progression. We therefore hypothesize that adenosine play a protective role in diabetic myocardial I/RI through the inhibition of calpain.

CaMKII inhibition mitigates ischemia/reperfusion-elicited calpain activation and the damage to membrane skeleton proteins in isolated rat hearts

Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been implicated in myocardial ischemia/reperfusion (IR) injury. The aim of this study was to determine the effect of CaMKII on the damage to membrane skeleton proteins, which is an important cause of IR injury. Isolated rat hearts were subjected to 45-min global ischemia/2-h reperfusion. Both KN-62 and KN-93 were used to inhibit CaMKII. Compared with controls, the hearts in the IR group exhibited remarkable myocardial injury area, LDH release, cell apoptosis and contractile dysfunction, along with an increase in the phosphorylation of CaMKII and its substrate phospholamban. Treatment with either KN-62 or KN-93 mitigated both the heart injury and the phosphorylation of CaMKII and phospholamban. The analysis of cell skeleton proteins revealed that IR injury resulted in an increase in the 150-kDa fragments resulting from the degradation of α-fodrin and dystrophin translocating from the sarcolemmal membrane to the cytosol and a decrease in the 220-kDa isoform of ankyrin-B. As expected, Evans blue dye staining showed an increase in membrane permeability or membrane rupture in the IR group. All of these alterations were alleviated by treatment with either KN-62 or KN-93. In addition, both KN-62 and KN-93 blocked the activity and membrane recruitment of calpain, a key protease responsible for destroying cell skeleton proteins during IR injury. In conclusion, our data provide evidence that damage to membrane skeleton proteins via calpain is a destructive downstream event of CaMKII activation in the setting of myocardial IR injury.

Glutamate protects against Ca2+ paradox-induced injury and inhibits calpain activity in isolated rat hearts

This study determined the effects of glutamate on the Ca2+ paradoxical heart, which is a model for Ca2+ overload‐induced injury during myocardial ischaemia and reperfusion, and evaluated its effect on a known mediator of injury, calpain. An isolated rat heart was retrogradely perfused in a Langendorff apparatus. Ca2+ paradox was elicited via perfusion with a Ca2+‐free Krebs‐Henseleit (KH) solution for 3 minutes followed by Ca2+‐containing normal KH solution for 30 minutes. The Ca2+ paradoxical heart exhibited almost no viable tissue on triphenyltetrazolium chloride staining and markedly increased LDH release, caspase‐3 activity, cytosolic cytochrome c content, and apoptotic index. These hearts also displayed significantly increased LVEDP and a disappearance of LVDP. Glutamate (5 and 20 mmol/L) significantly alleviated Ca2+ paradox‐induced injury. In contrast, 20 mmol/L mannitol had no effect on Ca2+ paradox. Ca2+ paradox significantly increased the extent of the translocation of μ‐calpain to the sarcolemmal membrane and the proteolysis of α‐fodrin, which suggests calpain activation. Glutamate also blocked these effects. A non‐selective inhibitor of glutamate transporters, dl‐TBOA (10 μmol/L), had no effect on control hearts, but it reversed glutamate‐induced cardioprotection and reduction in calpain activity. Glutamate treatment significantly increased intracellular glutamate content in the Ca2+ paradoxical heart, which was also blocked by dl‐TBOA. We conclude that glutamate protects the heart against Ca2+ overload‐induced injury via glutamate transporters, and the inhibition of calpain activity is involved in this process.

Myocardial Protective Effect of Urethane on Isolated Rat Hearts in Prolonged Hypothermic Preservation

BACKGROUND One of the most important factors restricting heart transplantation is the limited myocardial ischemia time. This study investigated the effects of urethane on the hypothermic preservation of donor rat hearts. MATERIALS AND METHODS Hearts isolated from rats were divided into 2 groups (n = 8), a control group with histidine-tryptophan-ketoglutarate (HTK) solution alone and an experimental group with HTK solution plus 30 mM urethane. Hearts were mounted on a Langendorff apparatus to estimate the baseline cardiac function; the hearts were then arrested and stored in one of the 2 solutions for 6 hours and 18 hours at 4 degrees C. After preservation, the hearts were reperfused, and cardiac function was evaluated. Lactate dehydrogenase (LDH) release, adenosine triphosphate (ATP) content, cardiomyocyte apoptosis, and myocardial ultrastructure were examined. RESULTS Compared with the control group, the experimental group showed a significantly higher recovery of cardiac function for both 6 hours and 18 hours of preservation and demonstrated a lower rate of cardiomyocyte apoptosis (8.5% + or - 1.2% versus 12.2% + or - 1.8% for 6 hours; 14.1% + or - 2.1% versus 31.4% + or - 2.7% for 18 hours). ATP content was significantly higher in the experimental group than in the control group after 18 hours of preservation (229.4 + or - 29.7 microg/g versus 153.2 + or - 21.1 microg/g). The experimental group also showed lower levels of LDH release after 18 hours of preservation. Electron microscopy studies demonstrated better cardiomyocyte structure in the experimental group for both 6 hours and 18 hours of preservation. CONCLUSIONS Use of urethane improved cardiac functional recovery and led to significant protective effects on rat hearts placed in a hypothermic preservation solution for a prolonged period.